Instruments in the lab

The labs are located at two sites: the Dept of Pharmaceutical Sciences, at the south campus of University at Buffalo, and the New York State Center of Excellence in Bioinformatics and Life Sciences (CEBLS, the main lab) in Buffalo, NY. These two campuses encompass a total of 1200 sq ft of well-equipped wet-lab devoted to cell culture, biochemistry experiments (for Western-blotting, electrophoresis, ELISA, etc.), column packing, treatment of proteomic samples and preparation for quantitative analysis. A Major equipment includes two ultra-centrifuges, one vacuum centrifuge, one lyophilizer, one set of solid phase purification system, one UV spectrometer, three hoods, one automatic cell counter and two incubators, as well as standard equipment for protein analysis.
There are two instrument rooms at 600 sq ft in total on the 3rd floor of CEBLS, which house a number of state-of-the-art the chromatography and mass spectrometer instruments, including:

  1. Three mass spectrometers: one high-resolution Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer, one ultra-high sensitivity Thermo Quantiva triple quadrupole mass spectrometer, and one Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer;
  2. Eight high performance liquid chromatography systems: one Eksigent nano-flow 2D-ultra system, one Eksigent nano-2D, one GE MDLC nano/micro chromatographic system, two Agilent 1200-series micro-flow HPLCs, one Waters UPLC, one Waters Bioseparation semi-preparative HPLC and one Shimadzu AC20 HPLC.

Among these instruments, the key ones are the Orbitrap Fusion and the Quantum Ultra EMR mass spectrometers. The Oribtrap Fusion provides high mass resolution (>120,000) and mass accuracy (<3 ppm). This instrument will provide highly confident identification and quantification of marker proteins in plasma, and is efficient to carry out the top-down proteomic analysis of low-MW proteins. The lab modified the injection strategy in the analyzer, which gained 20-fold more sensitivity than manufacturer setup. The ETD capacity provides optimal fragmentation for highly-charged peptides, which will improve the likelihood of identifying the target peptides. The Quantum Ultra EMR instruments are highly sensitive and able to decrease significantly the SRM chemical noise by using high-resolution monitoring. Therefore, this instrument will be used for selective, sensitive and accurate quantification of fragments from cTnI and target marker proteins in blood. Prior to MS analysis, samples will be chromatographically resolved on the nano-LC systems listed above, which provide hundreds of folds more sensitivity than by a conventional LC. The lab developed a highly efficient, robust and sensitive nano-spray interface (vented liquid junction) to couple the nano-LC system with different mass spectrometers.

UB Center for Computational Research
UB center for Computation Research (CCR) is located in the same building as the main laboratory. The CCR (www.ccr.buffalo.edu) is a leading academic supercomputing facility and maintains a high-performance computing environment and high-end visualization laboratories. Its technical staffs have broad expertise in scientific computing, numerical methods, software engineering, parallel processing, and visualization. The center's extensive computing facilities, which are housed in a state-of-the-art 4000 sq ft machine room, include a 2112 processor Intel Xeon-based (64-bit) Linux cluster, and a 64 processor shared memory Itanium-based cluster. CCR currently has more than 20 Tflops of aggregate computing capacity dedicated solely to support faculty led research projects at UB, and will soon undergo a $9M expansion to 50-70 Tflops of computing capacity (4000-6000 cores) and more than 800 Tbytes of high-performance storage capacity. CCR provides several layers of support for the life sciences/bioinformatics initiatives, including access to state-of-the-art hardware and software to facilitate research, the development of customized software/databases to support data analysis and workshops/training on software packages supported by CCR.
Currently the lab holds a 64-node concurrent license for SEQUEST, which is running on a Dell Linux cluster in the CCR. Both the above software and the hardware are dedicated to the lab. This will enable high-throughput analysis of the large amount of proteomic data that will be produced in this project.


Computer and software packages for data analysis
To extensively identify the myocardial proteins in swine plasma, the large amount of proteomic data will be analyzed both in the lab and in the UB CCR. In the lab, six high-end Dell 8-core work stations, as well as a high-performance data server are dedicated to data analysis. The primary software packages that we use and develop include: 1. Proteome Discoverer (Thermo Scientific) embedded with SEQUEST / MS-GF+, for protein identification; 2. Scaffold (Proteome Software), for post-search processing, protein filtering and grouping, and FDR control; 3. SIEVE (Thermo Scientific), for chromatogram alignment and global recgonition of MS1 quantitative features; 4. PEAKS (Bioinformatics Solutions Inc.), for De Novo sequencing; 5. Ingenuity Pathway Analysis (QIAGEN) for biological interpretation of bottom-up proteomic data; 6. in-house developed R scripts for data incorporation, quantification and bioinformatics analysis. We own licenses for all of the above mentioned software packages.

 

Update date: May, 2017